The discovery of the causative agent of hepatitis B together with the development of highly sensitive assays for the detection of the agent in donated blood, led to rapid virtual elimination of this virus from the blood supply. However other viral forms of transfusion-associated hepatitis soon became apparent, and have been termed generally Non-A Non-B hepatitis (NANBH). It is apparent from studies involving passaging of infected blood in chimpanzees that there exists more than one species of infective agent causative of NANBH (For example, see Bradley, et al., J. Infect. Dis., 148: 254 (1983)). It is also apparent from the early studies that isolation of pure virus would be extremely difficult because its presence in blood, even in the acute phases of infection, is on the order of only 1000 infective units per ml.
In spite of the failure of primary isolation, the virus was nevertheless characterized preliminarily by a number of criteria. Inactivation of the agent by chloroform implicated an envelope virus (Hotta & Evans, Virology, 2: 773 (1985)). Filtration of NANBH agents through membranes of different pore size indicated a size range of 30-60 nm. It was further determined that NANBH agents sedimented at approximately 200 S with a buoyant density of 1.24 g/cc. Finally, the agent was visualized by electron microscopy (Cabral, et al., Gastroent., 81: 120 (1981). All of the data are consistent with the conclusion that NANBH agents are toga or flaviviruses, or members of a group closely related to the toga or flaviviruses.
The breakthrough in NANBH research came in the course of passaging the NANBH virus, obtained from a contaminated purified Factor VIII, through chimpanzees. Bradley, et al., Seminars in Liver Disease, 6: 56 (1986) discovered that the titer of NANBH infectire units in one particular chimpanzee was a thousand fold greater than the titer ordinarily observed. This enriched source of NANBH agent permitted the creation and screening of a cDNA library by Houghton, et al (EP 0 318 216) utilizing the lambda gtll system devised by Young & Davis, PNAS, 80: 1194 (1983). EP 0 318 216 discloses a large open reading frame domain, which includes the C-100 polypeptide which is the target antigen in a commercial immunoassay for diagnosis of NANBH. This disclosure is supplemented in a second European Patent Application (EP 0 388 232) which discloses further sequences representing a sizable portion of the polypeptide possibly corresponding to the 5 prime end of the viral genome encoding putative structural proteins.
Other recent foreign applications include WO 90,02206 (Seto) which discloses further sequences cloned from liver sections of patients suffering from NANBH. WO 90/00597 (Neeley) presents a method of producing virus from immortalized human liver cells productive of active NANBH virus. An alternative method for preparing viral antigen is disclosed in EP 0 190 972 (Yohko) and involves a centrifugation procedure on sucrose gradients of proteins derived from infected chimpanzee liver tissue. Monoclonal antibodies were also obtained in '972 by transforming lymphocytes from NANBH infected humans or chimpanzees with Epstein-Barr virus, and screening for clones secreting antibody specific for the gradient purified antigens. The above NANBH polypeptide sequences are said to be useful in diagnostic assays.
Additional sequences for NANBH are set forth in EP 0 363 025 (Arima). The sequences were obtained from RNA extracted from viruses concentrated from approximately 100 liters of plasma from patients with NANBH. Sequences were identified by immunoscreening of a lambda gtll cDNA library. Of those clones screening positive, one such clone contained the clone 18 peptide having the amino acid sequence described in claim 2 formulae III in EP 0 363025 (Arima).